The present invention relates to methods for identifying compounds that bind RNAs. In particular, the present invention relates to methods for using a target RNA having a detectable label to screen a library of test compounds immobilized on one or more solid supports, and preferably, on one or more individual beads. Contacting a target RNA to a test compound can form a dye-labeled target RNA:support-attached test compound complex. Dye-labeled target RNA molecules in the complex label the test compounds attached to the solid supports, which can be separated from a plurality of unlabeled solid supports. Finally, the structure of the test compound attached to the solid support can be determined by decoding the solid support.
Protein-nucleic acid interactions are involved in many cellular functions, including transcription, RNA splicing, and translation. Readily accessible synthetic molecules that can bind with high affinity to specific sequences of single- or double-stranded nucleic acids have the potential to interfere with these interactions in a controllable way, making them attractive tools for molecular biology and medicine. Successful approaches for blocking binding sites on target nucleic acids include using duplex-forming antisense oligonucleotides (Miller, P. S. (1996) Progress in Nucl. Acid Res. and Mol. Biol. 52, 261-291) and peptide nucleic acids (xe2x80x9cPNAxe2x80x9d) (Nielsen, P. E. (1999) Current Opinion in Biotechnology 10, 71-75), which bind to nucleic acids via Watson-Crick base-pairing and using triplex-forming anti-gene oligonucleotides (Ping, Y.-H. et al. (1997) RNA 3, 850-860) and pyrrole-imidazole polyamide oligomers (Gottesfeld, J. M. et al. (1997) Nature 387, 202-205; White, S. et al. (1998) Nature 391, 468-471), which are specific for the major and minor grooves of a double helix, respectively. One advantage of using oligonucleotide-based classes of nucleic acid binding compounds is that they are easily identified by their primary or secondary structures. Another approach employs carbohydrate-based ligands, calicheamicin oligosaccharides, which interfere with the sequence-specific binding of transcription factors to DNA and inhibit transcription in vivo (Ho, S. N. et al. (1994) Proc. NatI. A cad. Sci. USA 91, 9203-9207; Liu, C. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 940-944).
Nucleic acids, and in particular RNA, may fold into complex tertiary structures consisting of local motifs such as loops, bulges, pseudoknots and turns (Chastain, M. and Tinoco, I., Jr. (19991) Progress in Nucleic Acid Res. and Mol. Biol. 41, 131-177; Chow, C. S. and Bogdan, F. M. (1997) Chemical Reviews 97, 1489-1514), which are critical for protein-RNA interactions (Weeks, K. M. and Crothers, D. M. (1993) Science 261, 1574-1577). The dependence of these interactions on the native three-dimensional structure of RNA makes it difficult to design synthetic agents using general, simple-to-use recognition rules analogous to those for the formation of double- and triple-helical nucleic acids. Since RNA-RNA and protein-RNA interactions may be important in, e.g., viral and microbial disease progression, it would be advantageous to have a general method for rapidly identifying compounds that bind to specific RNAs and that may prove to be antagonists of in vivo protein-RNA or RNA-RNA interactions.
Currently available methods for screening combinatorial compound libraries for compounds that bind to RNA are labor intensive and are not well-adapted to high throughput screening. Moreover, if a mixture of compounds is tested, using time-consuming deconvolution strategies may be necessary to identify the individual compounds in the mixture that have the most desirable properties (Hamy et al. (1997) Proc. NatI. Acad. Sci. USA 94, 3548-3553; Hamy et al. (1998) Biochemistry 37, 5086-5095). One frequently used method of identifying compounds that disrupt protein-RNA interactions is the gel mobility shift assay (Mei et al. (1998) Biochemistry 37, 14204-14212; Mei et al. (1997) Bioorganic and Medicinal Chem. 5:1173-84; Hamy et al. (1997) Proc. Nat. Acad. Sci. USA 94, 3548-3553; Hamy et al. (1998) Biochemistry 37, 5086-5095). In this assay, a protein/labeled RNA complex is formed, various concentrations of a potential inhibitor are added, and the resulting dissociation of the complex is monitored by observing the changing mobility of the labeled RNA on a gel. Although a dissociation constant for the potential inhibitor can be calculated using the assay, testing many compounds in this way is time consuming. Furthermore, additional experiments, such as RNase footprinting or NMR, may have to be performed to ensure that disruption of the complex is due to the compound binding to the RNA rather than to the protein (Hamy et al. (1997) Proc. NatI. Acad. Sci. USA 94, 3548-3553; Hamy et al. (1998) Biochemistry 37:5086-5095). Other methods for testing compounds for specific RNA binding include electrospray ionization mass spectrometry (ESI-MS), filter binding assays, and scintillation proximity assays (SPA) (Mei et al. (1998) Biochemistry 37, 14204-14212; Mei et al. (1997) Bioorganic and Medicinal Chem. 5:1173-84). Yet another method for screening for RNA binding compounds involves detecting changes in RNA conformation upon binding of the compound by, e.g., hybridization, treatment with conformation-specific nucleases, binding to matrices specific for single- or double-stranded nucleic acids or fluorescence resonance energy transfer (International Patent Publication WO 97/09342, published Mar. 13, 1997). The method described in WO 97/09342 has limited usefulness because not all compounds that bind to RNA will cause a detectable conformational change in the nucleic acid.
Screening of combinatorial libraries may also be performed using a computer. For example, databases of RNA three-dimensional structures can be examined to identify common xe2x80x9cmolecular-interaction sites,xe2x80x9d i.e., general structural motifs through which RNAs interact with other molecules, such as proteins. These molecular-interaction sites can then be used to computationally design compounds that bind at these sites. Virtual library compounds can be screened on the basis of their conformation, binding affinity, strain energy, solubility, and the like (Li (1998) DDT 3:105; Walters (1998) DDT 3:160). Once library compounds are selected, the compounds can be synthesized and tested. Although these methods are well-adapted to high-throughput screening of compounds, they are dependent on the accuracy of the RNA structures in the databases and on the ability of computer programs to accurately predict the conformations of potential inhibitors in solution.
Accordingly, there is clearly a need for fast and efficient methods for screening combinatorial compound libraries for molecules that bind to RNAs and potentially disrupt protein-RNA or RNA-RNA interactions.
Citation or identification of any reference in Section 2 of this application is not an admission that such reference is available as prior art to the present invention.
In a first embodiment, the present invention relates to a method for identifying a test compound that binds to a target RNA molecule, comprising the steps of: (a) contacting a dye-labeled target RNA molecule with substantially one type of test compound attached to a solid support, thereby providing a dye-labeled target RNA:support-attached test compound complex; and (b) determining the structure of the substantially one type of test compound of the RNA:test compound complex.
In a second embodiment, the present invention relates to a method for identifying a test compound that binds to a target RNA molecule, comprising the step of determining the structure of substantially one type of test compound of an RNA:test compound complex formed from contacting a dye-labeled target RNA molecule with substantially one type of test compound attached to a solid support.
In a third embodiment, the present invention relates to a method for forming a target RNA:test compound complex, comprising the step of contacting a target RNA molecule with the test compound identified from a method comprising the steps of: (a) contacting a dye-labeled target RNA molecule with substantially one type of test compound attached to a solid support, thereby providing a dye-labeled target RNA:support-attached test compound complex; and (b) determining the structure of the substantially one type of test compound of the RNA:test compound complex.
In a fourth embodiment, the present invention relates to a method for increasing or decreasing the production of a protein comprising the step of contacting a target messenger RNA molecule that encodes said protein with the test compound identified from a method comprising the steps of: (a) contacting a dye-labeled target RNA molecule with substantially one type of test compound attached to a solid support, thereby providing a dye-labeled target RNA:support-attached test compound complex; and (b) determining the structure of the substantially one type of test compound of the RNA:test compound complex.
In a fifth embodiment, the present invention relates to a method for treating or preventing a disease whose progression is associated with in vivo binding of a test compound to a target RNA, comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of the test compound, or a pharmaceutically acceptable salt thereof, identified according to a method comprising the steps of: (a) contacting a dye-labeled target RNA molecule with substantially one type of test compound attached to a solid support, thereby providing a dye-labeled target RNA:support-attached test compound complex; and (b) determining the structure of the substantially one type of test compound of the RNA:test compound complex.
In a sixth embodiment, the present invention relates to a method for treating or preventing HIV infection or AIDS in a patient, comprising administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound selected from the group consisting of:
H2N-(L)Lys-(D)Lys-(L)Asn-OH,
H2N-(L)Lys-(D)Lys-(D)Asn-OH,
H2N-(L)Lys-(L)Lys-(L)Asn-OH,
H2N-(L)Arg-(D)Lys-(L)Asn-OH,
H2N-(L)Arg-(D)Lys-(L)Val-OH,
H2N-(L)Arg-(D)Lys-(L)Arg-OH,
H2N-(D)Thr-(D)Lys-(L)Asn-OH, and
H2N-(D)Thr-(D)Lys-(L)Phe-OH